Cell自噬综述-To Be or Not to Be_ How Selective Autophagy and Cell Death Govern Cell Fate

Leading EdgeReviewTo Be or Not to Be?How Selective Autophagyand Cell Death Govern Cell FateDouglas R.Green1,*and Beth Levine2,*1Department of Immunology,St.Jude’s Children’s Research Hospital,Memphis,TN38205,USA2Center for Autophagy Research,Department of Internal Medicine,Department of Microbiology and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center,Dallas,TX75390,USA*Correspondence:douglas.green@(D.R.G.),beth.levine@(B.L.)/10.1016/j.cell.2014.02.049The health of metazoan organisms requires an effective response to organellar and cellular damage either by repair of such damage and/or by elimination of the damaged parts of the cells or the damaged cell in its entirety.Here,we consider the progress that has been made in the last few decades in determining the fates of damaged organelles and damaged cells through discrete, but genetically overlapping,pathways involving the selective autophagy and cell death machinery. We further discuss the ways in which the autophagy machinery may impact the clearance and con-sequences of dying cells for host physiology.Failure in the proper removal of damaged organelles and/or damaged cells by selective autophagy and cell death processes is likely to contribute to developmental abnormalities,cancer,aging,inflammation,and other diseases.IntroductionAs in all living things,each of our cells suffers the slings and arrows of outrageous fortune,facing damage from without and within.And,like the Prince of Denmark,each decides whether to be or not to be.To be,the cell must monitor and repair the damage.If not,it will‘‘melt,thaw,and resolve itself into a dew,’’dying and cleared from the body by other cells (with apologies to Shakespeare for scrambling his immortal words).Here,we consider how the molecular pathways of autophagy and cell death and,ultimately the clearance of dying cells,function in this crucial decision.Although autophagy and cell death occur in response to a wide variety of metabolic and other cues,our focus is restricted here to those aspects of each that are directly concerned with the quality control of cells—the‘‘garbage’’(cellular or organellar) that must be managed for organismal function.And although there are many important functions of quality control mecha-nisms(e.g.,DNA and membrane repair,cell growth and cell-cycle control,unfolded protein and endoplasmic reticulum (ER)stress responses,innate and adaptive immunity,and tumor suppression),our discussion is limited to the selective disposal of damaged or otherwise unwanted organelles and, when necessary,damaged or excess cells and how the autophagic and cell death mechanisms function in these pro-cesses.Overall,we focus on the overriding theme of waste management,but as we will see,many of the links between these elements remain largely unexplored.Further,although a great deal of what we know was delineated in yeast and invertebrate model systems,we largely restrict our consider-ation to what is known in mammals.Engaging AutophagyThe process of macroautophagy(herein,autophagy)is best understood in the context of nutrient starvation(Kroemer et al., 2010;Mizushima and Komatsu,2011).When energy in the form of ATP is limiting,AMP kinase(AMPK)becomes active, and this can drive autophagy.Similarly,deprivation from growth factors and/or amino acids leads to the inhibition of TORC1, which,when active,represses conventional autophagy.As a result of AMPK induction and/or TORC1inhibition,autophagy is engaged,although other signals may bypass AMPK and TORC1to engage autophagy(Figure1).The‘‘goal’’of the autophagy machinery is to deliver cytosolic materials to the interior of the lysosomes for degradation,thereby recovering sources of metabolic energy and requisite metabo-lites in times of starvation(general autophagy).Autophagy can similarly function to target damaged or otherwise unwanted organelles to lysosomes for removal(selective autophagy). Although we focus here primarily on selective autophagy,it is useful to also consider general autophagy to highlight similarities and distinctions between the two processes.In both cases a double-membrane structure,the auto-phagosome,fuses with lysosomes to deliver the contents for degradation,and this involves a proteolipid molecule,LC3-II,a component of the autophagosome composed of a protein, LC3,and a lipid,phosphatidylethanolamine.LC3-II is generated by a process resembling ubiquitination,involving E1,E2,and E3 ligases(Figure1).The parent molecule,LC3-I,is generated by the action of a protease,ATG4,which cleaves LC3to produce LC3-I.This is bound by the E1,ATG7,and transferred to the E2,ATG3.The E3ligase is a complex composed of ATG16L and ATG12-5;the latter is produced by another reaction inwhich Cell157,March27,2014ª2014Elsevier Inc.65ATG12is bound by the E1,ATG7,transferred to a different E2,ATG10,and from there to ATG5.The process by which ATG12-5is formed—and,subsequently,LC3-II (also known as LC3-PE)is generated—is referred to as the elongation reaction and is required for the formation of the autophagosome.Although not entirely understood,the generation of the LC3-coupled autophagosome appears to originate through extension of intracellular membranes,and several sources have been sug-gested,including ER,mitochondria,ER-mitochondrial contact sites,ER-Golgi intermediate compartment,the recycling endo-some,and the plasma membrane (Hamasaki et al.,2013).The initiation process requires the action of the Class III PI3kinase,VPS34,which converts phosphatidylinositol to phosphatylinosi-tol 3-phosphate (PI3P);this is the only enzyme that performs this function in cells (Meijer and Klionsky,2011).The activity ofVPS34requires VPS15(which is myristoylated and binds to membranes),a requisite regulator,Beclin 1,and other proteins,including ATG14L,that bind to Beclin 1.The complex,termed the initiation complex,generates PI3P,which dictates the site at which the double membrane subsequently elongates by the E1-E2-E3interaction described above (Figure 1).During starvation-induced autophagy,the function of the Beclin 1-VPS34initiation complex is controlled by a preinitiation complex that includes a protein kinase,ULK1,and the function of this kinase activates the initiation complex to generate PI3P (Russell et al.,2013).This preinitiation complex is,in turn,acti-vated by AMPK and inhibited by TORC1(Wirth et al.,2013),and as we have discussed,starvation conditions result in AMPK activation and TORC1inhibition.At least in the setting of glucose deprivation,AMPK can also act directly on theBeclinFigure 1.Overview of the General Autophagy PathwayCellular events and selected aspects of the molecular regulation involved in the lysosomal degradation pathway of autophagy in mammalian cells are shown.Several membrane sources may serve as the origin of the autophagosome and/or contribute to its expansion.A ‘‘preinitiation’’complex (also called the ULK complex)is negatively and positively regulated by upstream kinases that sense cellular nutrient and energy status,resulting in inhibitory and stimulatory phosphorylations on ULK1/2proteins.In addition to nutrient-sensing kinases shown here,other signals involved in autophagy induction may also regulate the activity of the ULK complex.The preinitiation complex activates the ‘‘initiation complex’’(also called the Class III PI3K complex)through ULK-dependent phosphorylation of key components and,likely,other mechanisms.Activation of the Class III PI3K complex requires the disruption of binding of Bcl-2anti-apoptotic proteins to Beclin 1and is also regulated by AMPK and a variety of other proteins not shown in the figure.The Class III PI3K complex generates PI3P at the site of nucleation of the isolation membrane (also known as the phagophore),which leads to the binding of PI3P-binding proteins (such as WIPI/II)and the subsequent recruitment of proteins involved in the ‘‘elongation reaction’’(also called the ubiquitin-like protein conjugation systems)to the isolation membrane.These proteins contribute to membrane expansion,resulting in the formation of a closed double-membrane structure,the autophagosome,which surrounds cargo destined for degradation.The phosphatidylethanolamine-conjugated form of the LC3(LC3-PE),generated by the ATG4-dependent proteolytic cleavage of LC3,and the action of the E1ligase,ATG7,the E2ligase,ATG3,and the E3ligase complex,ATG12/ATG5/ATG16L,is the only autophagy protein that stably associates with the mature autophagosome.The autophagosome fuses with a lysosome to form an autolysosome;inside the autolysosome,the sequestered contents are degraded and released into the cytoplasm for te endosomes or multivesicular bodies can also fuse with autophagosomes,generating intermediate structures known as amphisomes,and they also contribute to the formation of mature lysosomes.Additional proteins (not depicted in diagram)function in the fusion of autophagosomes and lysosomes.The general autophagy pathway has numerous functions in cellular homeostasis (examples listed in box labeled ‘‘physiological functions’’),which contribute to the role of autophagy in development and protection against different diseases.66Cell 157,March 27,2014ª2014Elsevier Inc.1/VPS34complex(Kim et al.,2013a).For starvation-induced autophagy,it is also necessary to unleash negative regulators of the Beclin1-VPS34initiation complex,such as Bcl-2/Bcl-x L (Wirth et al.,2013).Selective Autophagic Removal of OrganellesAs discussed in the Introduction,autophagy can function to re-move damaged or otherwise unwanted organelles in a cell.By ‘‘unwanted,’’we mean organelles that are removed during differ-entiation(e.g.,in maturing erythrocytes)or when environmental factors(e.g.,hypoxia)disfavor some organelles in the cell.We refer to this process as selective autophagy.When considering selective autophagy,we are faced with two problems.First, how does the process‘‘know’’which structures or organelles to target for removal?And second,how does this occur even when the conventional autophagy machinery is suppressed(at least partially),such as in nutrient-rich conditions?With regard to the latter,the problem is confounded by the simple fact that lysosomal digestion of organelles will itself provide amino acids and other metabolites,presumably activating TORC1and sup-pressing AMPK.As we have seen,such conditions inhibit the function of the preinitiation complex.Nevertheless,animals lack-ing Ulk1display a defect in at least one selective autophagic pro-cess,that of efficient removal of mitochondria during erythrocyte development(Kundu et al.,2008).Presumably,there are ways to bypass conventional inhibitory mechanisms to engage Ulk1 activity and promote selective autophagy in some settings.Alter-natively,the preinitiation complex may be bypassed in some situations.We will not fully resolve this paradox here but perhaps provide clues as we consider thefirst problem—how specific cargoes are marked for clearance.Before considering this issue,it may be useful to note that, even in nutrient-starved conditions,autophagy may be selective. Ribosomes represent a major portion of the biomass of many cells,and upon starvation,these are more rapidly removed than other structures in the cell(Cebollero et al.,2012).Similarly, there appears to be selective removal of peroxisomes during starvation(Hara-Kuge and Fujiki,2008).The same may be the case for ER(reticulophagy),although it remains possible that, in this case,this is a consequence of developing the requisite autophagosomes for nutritional supplementation using the ER membrane(see above).Another possible selection during star-vation is the preservation of functional mitochondria;because these are necessary for the catabolism of free fatty acids or amino acids and for the optimal generation of energy from glucose (all generated by lysosomal digestion),it simply does not make sense that inadvertent removal of mitochondria during starvation would be permitted.Such possible‘‘antiselection,’’however,has not been fully documented or adequately explored.Targeting in selective organellar autophagy is perhaps best analyzed in the clearance of mitochondria(mitophagy)and per-oxisomes(pexophagy).Tissues or cells lacking requisite compo-nents of the autophagy elongation machinery(e.g.,ATG5and ATG7)often display greatly increased numbers of apparently damaged mitochondria(Mizushima and Levine,2010)and per-oxisomes(Till et al.,2012).That said,there is evidence that, even in the absence of ATG5and ATG7,some selective mitoph-agy continues via unknown mechanisms,perhaps via vesicular trafficking between mitochondria and lysosomes(Soubannier et al.,2012).Nevertheless,accumulated observations indicate that the autophagy elongation machinery and autophagosome formation are important for selective autophagy of damaged or otherwise unwanted organelles.One way in which damaged mitochondria are removed by autophagy involves the action of two proteins,PINK1and Parkin (Figure2).PINK1is a kinase that is constitutively imported into functional mitochondria and degraded by the rhomboid prote-ase,PARL.As with most mitochondrial import,this requires the transmembrane potential of the inner mitochondrial mem-brane,DJ m.Loss of this potential,which can occur when the electron transport chain is damaged or if protons are allowed to pass freely across the inner membrane(i.e.,due to the expres-sion of uncoupler protein[UCP],presence of environmental pro-tonophores,or as a consequence of the mitochondrial perme-ability transition)causes active PINK1to accumulate on the cytosolic face of the outer mitochondrial membrane.This then recruits and activates Parkin,which is a ubiquitin E3-ligase, which then ubiquitinates proteins on the mitochondria. Although it is not clear how ubiquitination triggers mitophagy, several ubiquitin-binding proteins bear a motif that binds to LC3. These include p62/sequestosome1,NBR1,and optineurin,and the binding of p62to LC3has been implicated in mitophagy in some studies(Shaid et al.,2013).This leads to the notion that it is the binding of LC3to ubiquitinated mitochondrial proteins that focuses the autophagy machinery on mitophagy(and perhaps other forms of selective autophagy).However,this idea has been challenged by studies showing that p62is not required for Parkin-mediated mitophagy(Narendra et al., 2010).Moreover,AMBRA1(a positive regulator of the Beclin1/ Class III PI3K initiation complex)binds to Parkin during mitoph-agy(Van Humbeeck et al.,2011),and several autophagy pro-teins,including ULK1,ATG14,ATG16L,and ATG9,are recruited to depolarized mitochondria in Parkin-expressing cells indepen-dently of LC3(Itakura et al.,2012).This suggests a model in which the damaged mitochondrion is not‘‘recognized’’by a pre-formed isolation membrane but,rather,recruits the machinery necessary for the de novo formation of an autophagosome. Although it is understood that the activation of PINK1and Parkin can trigger mitophagy,it is also likely that mitophagy proceeds via other mechanisms(Figure2).One such mechanism involves either of two related proteins,BNIP3and NIX(also known as BNIP3L).Animals lacking NIX fail to efficiently clear mitochondria in maturing erythrocytes,leading to anemia(San-doval et al.,2008),an effect not observed in Parkin-deficient animals.Similarly,during hypoxia,BNIP3expression is induced by HIF1,promoting mitophagy(Zhang et al.,2008).These pro-teins may act to directly recruit the autophagy machinery to the mitochondria(Zhang and Ney,2009)and may also promote mitophagy through interaction with Bcl-2,an antiapoptotic pro-tein(see below)that resides on the mitochondrial outer mem-brane.Both BNIP3and NIX promote the release of Beclin1 from Bcl-2,and this may,in turn,promote mitophagy,although the details are unclear.Another mitochondrial outer membrane protein,FUNDC1,may also be required for mitophagy through interaction with LC3in a process regulated by hypoxia and FUNDC1dephosphorylation(Liu et al.,2012).Cell157,March27,2014ª2014Elsevier Inc.67Although pieces of the puzzle are beginning to emerge,a comprehensive understanding of how specific cargoes are targeted for autophagic degradation is still lacking.Clearly,the model of ubquitinated cargo binding to LC3-interacting proteins (through their ubiquitin-binding domains),which subsequently bind to LC3(through their LC3-interaction region (LIR)motifs),and thereby targeting the cargo for autophagy is—at least in many cases—an oversimplification.Even in this model,the rele-vant substrates for ubiquitination,the spectrum of ubiquitin ligases,the spectrum of ubiquitin-binding LC3-interacting auto-phagy ’’receptor’’proteins,and the signals that trigger the initial ubiquitination of relevant substrates are not completely known.In the future,we must look beyond established paradigms for additional signals and mechanisms of selective autophagic removal of damaged organelles.One interesting alternative possibility is based on changes in lipid exposure,at least in the context of mitophagy.Damaged mitochondria expose cardiolipin,which is normally present pre-dominantly on the inner mitochondrial membrane,on the outer membrane (Chu et al.,2013).Vertebrate LC3orthologs bind to cardiolipin,and this binding appears to be involved in the selec-tive removal of the damaged mitochondria.If so,this may be specific to mitophagy in vertebrates,as the requisite docking site is not conserved in invertebrate or yeast orthologs of LC3.Although,in this discussion,we have focused on the selective removal of damaged organelles,we note that insights can be gleaned from examination of the process of xenophagy,the selective removal of infectious intracellular organisms by auto-phagic processes (Deretic et al.,2013),and this may inform our model further.For example,it has been shown that ATG16L is recruited to ubiquitin upstream of LC3lipidation in xenophagic removal (Fujita et al.,2013),suggesting that ubiqui-tinated proteins on damaged organelles are potentially targeted by the ATG5-12-ATG16L E3ligase to direct the process.Of note,although lipid droplets are technically not organelles,their clearance is important for maintaining cellular health,andFigure 2.Roles of Autophagy Proteins in the Removal of Unwanted Organelles and in the Removal of CellsThe left panel shows Parkin-dependent and Parkin-independent mechanisms involved in the selective degradation of mitochondria by autophagy (mitophagy).In Parkin-dependent mitophagy,mitochondrial damage and loss of mitochondrial membrane potential (DJ m)lead to localization of the kinase,PINK1,on the cytoplasmic surface of the mitochondria,resulting in recruitment of the E3ubiquitin ligase,Parkin,to the mitochondria,followed by the ubiquitination of mito-chondrial proteins and the formation of an isolation membrane that surrounds the damaged mitochondria.In Parkin-independent mitophagy,proteins such as Nix (shown in figure),BNIP3,and FUNDC1(not shown in the figure)bind to LC3.Other autophagy proteins may be involved in Parkin-dependent and Parkin-independent mitophagy (discussed in the text).The precise details of how an isolation membrane is formed around specific mitochondria earmarked for degradation are unclear.Other damaged/unwanted organelles such as ER,peroxisomes,and lipid droplets can also be degraded by selective autophagy;the molecular mechanisms of these forms of selective autophagy are not well understood in mammalian cells.The right panel depicts roles of LAP of apoptotic corpses and of live cells (entosis).In LAP,components of the autophagy initiation complex (Beclin 1and VPS34)are recruited to the phagosome,which leads to recruitment of LC3-PE and facilitation of phagolyosomal fusion.This process requires other components of the elongation machinery,but—in contrast to general autophagy or selective autophagy—proceeds independently of the ULK preinitiation complex.68Cell 157,March 27,2014ª2014Elsevier Inc.their removal by selective autophagy(lipophagy)may bear some similarities to the selective removal of damaged organelles(Liu and Czaja,2013).Lipophagy is a documented alternative form of lipid metabolism,and its failure in animals lacking efficient general or selective autophagy factors can lead to hepatic accumulation of intracellular lipid droplets(Singh et al.,2009; Orvedahl et al.,2011).However,the precise mechanisms by which lipid droplets are targeted for autophagic removal are underexplored.Consequences of Defective Selective Organellar AutophagyIt is self-evident that the selective removal of damaged or excess organelles is a critical homeostatic process,but beyond this,our information on what happens when this goes wrong is somewhat limited.There is an accumulation of damaged organelles (including mitochondria,perixosomes,and ER)and organ degeneration in mice with tissue-specific knockout of core autophagy genes such as Atg5and Atg7in liver,neurons,heart, pancreatic acinar cells,muscle,podocytes,adipocytes,and he-matopoietic stem cells(Mizushima and Levine,2010).Although it may not be possible to dissociate the effects of general autophagy from those of selective autophagy,it is reasonable to postulate that these phenotypes are partly related to defects in selective organellar autophagy,and at a minimum,such studies unequivocally establish a role for autophagy genes in the removal of damaged organelles in vivo.The proper removal of excess or unwanted mitochondria is likely necessary for certain key aspects in development.As dis-cussed above,the mitophagy factor Nix is required for mito-chondrial clearance during erythroid maturation in vivo(San-doval et al.,2008),and mouse erythrocytes lacking general autophagy factors such as Ulk1and Atg7fail to clear mitochon-dria(Kundu et al.,2008;Mortensen et al.,2010).Reduction in mitochondrial number may also contribute to the role of core autophagy genes,such as Atg7,in white adipocyte differentia-tion(Zhang et al.,2009).An intriguing question is whether selec-tive mitophagy—of paternal mitochondria—during embryonic development underlies mammalian maternal mitochondrial DNA(mtDNA)inheritance(Levine and Elazar,2011).In C.elegans,several studies showed that paternal mitochondria and mtDNA are eliminated from the fertilized oocyte by auto-phagy(with surrounding membranous organelles,but not the mitochondria themselves,marked by ubiquitin)(Al Rawi et al., 2011;Sato and Sato,2011;Zhou et al.,2011).In one of these studies(Al Rawi et al.,2011),p62and LC3were also found to colocalize with sperm mitochondria after fertilization in mice. However,a more recent study confirmed that sperm mitochon-dria colocalized with p62and LC3in mouse embryos but concluded that this was not involved in their degradation(Luo et al.,2013).Thus,the question of whether selective mitophagy explains why our mitochondrial DNA comes mainly from our mothers remains to be resolved.An emerging far-reaching biomedical paradigm is that defects in mitophagy—presumably through resulting abnormal mito-chondrial function,abnormal mitochondrial biogenesis,and/or increased mitochondrial generation of reactive oxygen species (leading to genomic instability and enhanced proinflammatory signaling)—contribute to cancer,neurodegenerative diseases,myopathies,aging,and inflammatory disorders(reviewed in Ding and Yin,2012;Green et al.,2011;Lu et al.,2013;Narendra and Youle,2011).This paradigm intuitively makes sense and is consistent with a large body of literature in autophagy-deficient mice.Yet,it is difficult to establish a direct causal relationship between mitophagy defects and disease in mice lacking general autophagy factors.Presumably,phenotypes observed in mice lacking selective mitophagy factors may be more informative. For example,Parkin-deficient mice have cancer-prone pheno-types,including accelerated intestinal adenoma development (in the background of Apc mutation)(Poulogiannis et al.,2010) and the development of hepatocellular carcinoma(Fujiwara et al.,2008).However,these studies also do not provide direct evidence that Parkin-mediated mitophagy,rather than other potential effects of Parkin,contribute to its role in tumor suppres-sion.Moreover,mice lacking Ulk1(Kundu et al.,2008)or Nix (Sandoval et al.,2008)have progressive anemia with mature erythrocytes containing mitochondria but no other obvious can-cer-prone defects.In addition,Parkin-null mice clear defective mitochondria normally in dopaminergic neurons in the substantia nigra(Sterky et al.,2011),even though PARKIN and PINK1mu-tations in humans lead to overt degeneration of these neurons and Parkinson’s disease.It is not unlikely that there are several overlapping mechanisms for selective autophagy that compen-sate for such deficiencies.Another possible explanation for the lack of more striking phenotypes in mice lacking selective autophagy factors is that other processes help to mediate the damage that should accrue when damaged organelles are not effectively cleared from cells,including perhaps the removal of the cells themselves,which is considered next.Removing Excess or Damaged CellsIn multicellular organisms,the death of a cell is usually easily tolerated and,indeed,part of normal development and homeo-stasis.Dying cells,regardless of the mode of cell death,are rapidly cleared from the body,either by shedding(e.g.,skin and mucosa)or removal by phagocytosis(Figure2).The latter in mammals is usually by‘‘professional’’phagocytes such as macrophages but can be mediated by other cells such as epithelia.For our discussion,it is useful to consider several distinctions between the many ways that cells can die(Green,2011).First, cells can die actively or passively,that is,molecularly partici-pating in their own demise or not.Passive cell death occurs when a cell has accumulated so much acute damage that it cannot maintain its plasma membrane and usually dies by a pro-cess that by morphology is classified as necrosis.Necrotic cells swell as water enters,expanding organelles and the nucleus (as we will see,however,morphological necrosis can also be active).Passive cell death can occur by environmental insult or by‘‘murder,’’such as via the action of complement,cytotoxic lymphocytes,or active engulfment of a living cell(e.g.,when aged erythrocytes are cleared or when epithelial cells lose con-tact with basal lamina).In contrast,active cell death involves intracellular processes that must be engaged for the cell to die. This is often referred to as‘‘programmed cell death,’’although the term was originally coined to indicate those cells that die at a defined(programmed)time in development.Here,we do notCell157,March27,2014ª2014Elsevier Inc.69distinguish between active and programmed,and we use both to refer to those cell deaths that involve the participation of intra-cellular molecular machinery.Active cell death can further be parsed into two general cate-gories:cell suicide and cell sabotage (Green and Victor,2012).Cell sabotage is akin to the conventional meaning of sabotage:just as a train must be moving quickly if it is to undergo destruc-tion when a rail is dislodged,some disruptions of cellular func-tions may derail a cell to its destruction only if these functions are actively engaged.In cellular sabotage,the death is therefore molecularly active,but the process (presumably)was not selected during the evolution of multicellularity to transduce the death signal.Examples of cellular sabotage likely include the recently identified process of ferroptosis (Dixon et al.,2012),overproduction of reactive oxygen species,and perhaps some forms of mitotic catastrophe.Cellular suicide,in contrast,involves the engagement of evolu-tionarily selected (again,presumably)molecular pathways that result in cell death (Figure 3).This is best exemplified by the process of apoptosis.There are multiple pathways of apoptosis,all of which involve the activation of caspase proteases,which cleave many substrates within the cell,leading to its demise.Apoptosis is formally defined by morphology,in which the cell shrinks,the nucleus condenses,and the cell often fragments into smaller membrane-bound bodies,although more recent de-scriptions rely on the detection of caspase activation (Galluzzi et al.,2012).In most apoptotic pathways,caspase activation oc-curs by the formation of ‘‘caspase activation platforms’’that bind and activate monomeric initiator caspases (e.g.,caspase-8and caspase-9),which then cleave and thereby activate the execu-tioner caspases (caspase-3and caspase-7)to orchestrate the death of the cell.Although apoptosis occurs in a variety of settings and often via different pathways,our focus on the clearance of damaged and excess cells focuses much of our attention on only one such pathway that functions in this regard.In this,the mitochon-drial (or intrinsic)pathway of apoptosis,the proteins of the Bcl-2family control a process whereby the outer membranes of mito-chondria become permeable (mitochondrial outer membrane permeabilization,MOMP),releasing the proteins of the inter-membrane space to the cytosol.Cytochrome c,thus released,activates apoptosis-activating factor-1(APAF1)to form a caspase activation platform for the activation of the initiator caspase,caspase-9(Figure 3).Figure 3.Cell Death Pathways Engaged by Cellular DamageCellular damage induces cell death by inducing expression and/or modification of proapoptotic BH3-only proteins of the Bcl-2family (inset),which engage the mitochondrial pathway of apoptosis,in which MOMP releases proteins of the mitochondrial intermembrane space.Among these is cytochrome c,which activates APAF1to form a caspase-activation platform (the apoptosome)that binds and activates caspase-9.This then cleaves and thereby activates execu-tioner caspases to promote apoptosis.Cellular damage can also induce the expression of death ligands of the TNF family,which bind their receptors to promote the activation of caspase-8by FADD.The latter is antagonized by expression of c-FLIP L ,and the caspase-8-FLIP heterodimer does not promote apoptosis but instead blocks another cell death pathway engaged by death receptors,necroptosis.Necroptosis involves the activation of RIPK1and RIPK3,resulting in phosphorylation and activation of the pseudokinase,MLKL,which promotes an active necrotic cell death.70Cell 157,March 27,2014ª2014Elsevier Inc.。